This proposal describes a five year training plan with the goal of developing the candidate into an independent investigator. The principal investigator will expand the scientific background and skills she has already attained through her research activities in the laboratory of Dr. Fred Gorelick, an expert in the molecular mechanisms of pancreatitis. By implementing a structured research, training, and career development plan under the mentorship of Dr. Gorelick and Dr. Michael Nathanson, an expert in calcium signaling in digestive epithelia, the candidate will gain the analytic framework, technical abilities, and knowledge base to succeed as an independent physician-scientist. Yale University and the Section of Digestive Diseases, with their rich network of diverse faculty and ample biomedical facilities, provide an ideal environment to pursue this plan. The pathogenesis of acute pancreatitis, a common and life-threatening disease, is incompletely understood. Clinical conditions characterized by acute acid loads predispose to the development of acute pancreatitis. Recently, the Gorelick lab has shown that low pH sensitizes to pancreatitis responses in vitro and in vivo. However, the mechanisms responsible for this sensitization are unknown. This proposal hypothesizes that low pH exerts its injurious effects by transforming intracellular calcium signaling and gap junctional intercellular communication in acinar cells from a physiologic pattern to one that causes pancreatitis. The specific aims of this proposal are to: 1) Determine the effects of low pH on intracellular calcium signaling in the acinar cell 2) Determine the effects of low pH on intercellular communication and 3) Determine whether acid-induced pancreatitis responses are mediated by pathologic calcium signals and/or changes in gap junctional intercellular communication. To establish the effects of low pH on calcium signals, isolated acinar cells will be examined through confocal microscopy. Functional aspects of gap junctional communication will be investigated by quantifying the degree of calcium signal synchrony and by examining dye transfer through gap junctions. Structural aspects of gap junctional intercellular communication will be examined by characterizing the degradation patterns of the predominant gap junction protein, connexin32. Finally, the role of calcium signals and gap junctional communication in acid-induced injury will be examined using in vitro and in vivo models of pancreatitis. Our preliminary data indicates that low pH decreases oscillation frequency and increases oscillation amplitude in isolated acini treated with physiologic concentrations of the cholecystokinin orthologue, cerulein. Additionally, low pH inhibits intercellular communication and leads to decreased levels of connexin32. Finally, we have found that inhibition of the ryanodine receptor, which mediates both the changes in calcium signaling and intercellular communication, reduces acid-induced zymogen activation and cellular injury in vitro. By establishing a link between harmful effects of acidemia and intracellular calcium signaling and intercellular gap junctional communication in the acinar cell, therapeutic strategies to treat or prevent acidosis-associated pancreatitis could be developed. PUBLIC HEALTH RELEVANCE: Acute pancreatitis is a common, potentially life-threatening disease of the pancreas, for which there is no effective treatment. Acid loads, seen in severe infections, diabetic complications, and many other clinical conditions can predispose to the development of acute pancreatitis. This proposal seeks to understand the mechanisms underlying the injurious effects of acid on the pancreas so that therapeutic strategies to treat or prevent acid-associated pancreatitis can be developed.